Association of Lipopolysaccharide-Toll-Like Receptor 4 Signaling and Microalbuminuria in Patients with Type 2 Diabetes Mellitus

Figures & data

Table 1 Clinical and Laboratory Characteristics of the Participants

Parameter	Control Group (n = 28)	T2DM Group (n = 32)	T2DM+Microalbuminuria Group (n = 29)	P value
Age (years)	51.86±5.39	50.41±10.38	58.38±11.89 ^c	0.005
Sex (male, %)	14 (50.00%)	19 (59.38%)	16 (55.17%)	0.767
T2DM duration (years)	–	1.50 (0.10, 7.75)	8.00 (2.00, 13.50) ^c	0.007
Smoking, n (%)	5 (17.86%)	10 (31.25%)	5 (17.24%)	0.331
Alcohol consumption, n (%)	7 (25.00%)	13 (40.62%)	8 (27.59%)	0.370
BMI (kg/m^2)	23.72±2.44	24.65±3.32	24.68±3.05	0.384
SBP (mmHg)	120.25±12.60	125.69±16.02	125.10±25.72	0.489
DBP (mmHg)	76.79±5.92	79.06±9.80	79.69±8.80	0.395
FBG (mmol/L)	5.35±0.81	10.27±3.41 ^b	11.54±3.83 ^b, c	<0.001
HbA1c (%)	5.75±0.31	9.99±2.02 ^b	10.14±2.36 ^b, c	<0.001
HOMA-IR	1.42 (0.97, 2.29)	3.12 (1.76, 4.14) ^b	3.23 (1.97, 4.42) ^b, c	<0.001
TC (mmol/L)	4.79±0.77	5.04±0.84	4.53±1.15	0.118
TG (mmol/L)	1.28 (1.00, 1.61)	1.74 (1.22, 2.59)	1.43 (1.19, 2.11)	0.044
HDL (mmol/L)	1.48±0.55	1.19±0.24 ^b	1.16±0.22 ^b	0.002
LDL (mmol/L)	3.07±0.78	3.65±0.77 ^b	3.17±1.10	0.027
Cr (µmol/L)	66.00±13.70	56.66±11.90	61.65±15.97	0.036
Ccr (mL/min)	123.98±40.96	111.31±32.84	114.86±41.70	<0.001
Urinary albumin (mg/24h)	2.17 (0.82,12.30)	10.61 (3.63, 15.08) ^b	65.52 (40.39, 154.18) ^b, c	<0.001
UAER (μg/min)	1.51 (0.57, 9.48)	7.36 (2.52, 10.48) ^b	45.50 (29.04, 107.07) ^b, c	<0.001
TLR4 (%)	10.48±2.10	12.72±3.34 ^b	14.23±5.25 ^b, c	0.001
IL-6 (pg/mL)	4.00 (3.33, 5.48)	3.30 (2.73, 4.20)	5.90 (4.15, 9.25) ^b, c	<0.001
hs-CRP (mg/dL)	0.35 (0.20, 0.70)	0.85 (0.20, 1.50) ^b	2.30 (0.75, 3.95) ^b, c	<0.001
DLA (ug/mL)	3.44±0.97	4.52±1.66 ^b	5.02±1.83 ^b, c	0.001
DAO (ng/mL)	231.28±49.89	267.32±79.83 ^b	299.99±67.55 ^b, c	0.001
LPS (pg/mL)	583.26±198.56	748.40±339.94 ^b	1212.47±422.15 ^b, c	<0.001

Notes: All values are expressed as mean ± SD, median value (interquartile range), or n (%); ^bP < 0.05 vs control group; ^c P < 0.05 vs T2DM group.

Abbreviations: T”DM, type 2 diabetes mellitus; Cr, creatinine; UAER, urinary albumin excretion rate; BMI, body mass index; SBP, systolic blood pressure; DAO, diamine oxidase; DBP, diastolic blood pressure; FBG, fasting blood glucose; HbA1c, glycosylated hemoglobin A1c; LPS, lipopolysaccharide; HOMA-IR, homoeostasis model assessment-insulin resistance; TC, cholesterol; TLR4, toll-like receptor 4; TG, triglyceride; HDL, high-density lipoprotein; LDL, low-density lipoprotein; Ccr: creatinine clearance; IL-6, interleukin-6; DLA, d-lactic acid; hs-CRP, high-sensitivity C-reactive protein.

Figure 1 TLR4 expression in peripheral blood mononuclear cells (PBMCs) (A) negative control, (B) control, (C) T2DM, and (D) T2DM+microalbuminuria groups. The percentage of double-positive cells stained with antibodies against CD14 and TLR4 was assessed by flow cytometry. The blank control was a tube without fluorescent antibodies.

Table 2 Correlations of Serum LPS with Clinical and Laboratory Characteristics

	Age	Duration	FBG	HOMA-IR	HbA1c	HDL	hs-CRP	IL-6	UAER	DLA	DAO	TLR4
rho	0.242	0.480	0.420	0.380	0.372	−0.214	0.281	0.248	0.556	0.594	0.663	0.251
P value	0.022	0.000	0.000	0.000	0.000	0.044	0.008	0.019	0.000	0.000	0.000	0.018

Abbreviations: LPS, lipopolysaccharide; FBG, fasting blood glucose; UAER, urinary albumin excretion rate; HbA1c, glycosylated hemoglobin A1c; HOMA-IR, homoeostasis model assessment-insulin resistance; DLA, d-lactic acid; HDL, high-density lipoprotein; TLR4, toll-like receptor 4; IL-6, interleukin 6; DAO, diamine oxidase; hs-CRP, high-sensitivity C-reactive protein.

Table 3 Correlations of UAER with Clinical and Laboratory Characteristics

	Age	Duration	FBG	HOMA-IR	HbA1c	HDL	hs-CRP	IL-6	LPS	DLA	DAO	TLR4
rho	0.193	0.597	0.481	0.271	0.450	−0.279	0.333	0.322	0.556	0.270	0.294	0.291
P value	0.070	0.000	0.000	0.010	0.000	0.008	0.001	0.002	0.000	0.010	0.005	0.006

Abbreviations: UAER, urinary albumin excretion rate; TLR4, toll-like receptor 4; LPS, lipopolysaccharide; FBG, fasting blood glucose; hs-CRP, high-sensitivity C-reactive protein; HDL, high-density lipoprotein; HbA1c, glycosylated hemoglobin A1c; DLA, d-lactic acid; HOMA-IR, homoeostasis model assessment-insulin resistance; IL-6, interleukin-6; DAO, diamine oxidase.

Table 4 Correlations of TLR4 Expression with Clinical and Laboratory Characteristics

	Age	Duration	FBG	HOMA-IR	HbA1c	HDL	hs-CRP	IL-6	UAER	LPS	DLA	DAO
rho	0.120	0.334	0.229	0.231	0.224	−0.012	0.272	0.041	0.291	0.251	0.220	0.020
P value	0.261	0.001	0.031	0.029	0.035	0.909	0.010	0.706	0.006	0.018	0.039	0.853

Abbreviations: TLR4, toll-like receptor 4; LPS, lipopolysaccharide; FBG, fasting blood glucose; hs-CRP, high-sensitivity C-reactive protein; HbA1c, glycosylated hemoglobin A1c; DAO, diamine oxidase; HOMA-IR, homoeostasis model assessment-insulin resistance; DLA, d-lactic acid; HDL, high-density lipoprotein; UAER, urinary albumin excretion rate; IL-6, interleukin-6.

Table 5 Multivariate Stepwise Linear Regression Analysis of Factors Independently Associated with UAER

Parameter	β	95% CI	t	P value
LPS	0.041	0.010─0.072	2.651	0.010
HbA1c	5.419	0.046─10.792	2.008	0.048
TLR4	2.401	0.097─4.705	2.074	0.041
hs-CRP	6.211	1.355─11.068	2.546	0.013

Abbreviations: UAER, urinary albumin excretion rate; LPS, lipopolysaccharide; HbA1c, glycosylated hemoglobin A1c; TLR4, toll-like receptor-4; DLA, d-lactic acid; hs-CRP, high-sensitivity C-reactive protein; CI, confidence interval.

Table 6 Binary Logistic Regression Analysis of Risk Factors for Microalbuminuria in T2DM

Parameter	Odds Ratio	95% CI	P value
FBG	1.415	1.045–1.918	0.024
hs-CRP	1.984	1.264–3.122	0.003
TLR4	1.254	1.010–1.557	0.041
LPS	1.012	1.004–1.018	0.000

Abbreviations: T2DM, type 2 diabetes mellitus; DLA, d-lactic acid; FBG, fasting blood glucose; TLR4, toll-like receptor 4; CI, confidence interval; hs-CRP, high-sensitivity C-reactive protein; LPS, lipopolysaccharide.